JPS6354692B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an anti-coccidial agent capable of immunologically preventing chicken coccidiosis and a method for inoculating the anti-coccidial agent. Coccidiosis of chickens is an infectious disease caused by infection with a certain type of parasitic protozoan and is widely distributed around the world. Infected animals exhibit symptoms such as diarrhea and bloody stools, and often die if treatment is delayed or the symptoms are severe. Coccidiosis has been considered the most damaging disease for poultry farmers because it inhibits growth even if it does not lead to death. Conventionally, drugs including vaccines have been orally administered to prevent coccidiosis. A coccidiosis vaccine is a mixture of several types of oocysts that are susceptible to coccidiosis, and this is orally administered to chickens. but,
Prophylaxis methods using such drugs have problems such as a decline in effectiveness due to the acquisition of drug resistance, and prophylaxis methods using vaccines have the disadvantage that the duration of immunity is short and cannot provide complete protection even during the period of immunity. Ta. The present inventors conducted research to solve the above problem and found that coccidiosis can be prevented by inoculating sporozoites of Eimeria tenella, one of the protozoa of chicken coccidiosis, through the cloaca of chickens. I found out. The sporozoite referred to in the present invention is a form of the growth ring of the oocyst of Eimeria tenella, and refers to a living sporozoite. An example of the method for preparing this sporozoite is shown below. Sporocysts are obtained by grinding the membranes of mature oocysts of Eimeria tenella isolated from the feces, cecal wall, and cecal contents of chickens infected with coccidiosis caused by Eimeria tenella. Next, the sporocysts are treated with chicken bile and trypsin to excyst the sporozoites. The material thus obtained mainly contains sporozoites, but also contains oocysts and sporocysts, as well as oocysts and sporocyst membranes. The sporozoites are then inoculated into chickens. The inoculation method involves suspending sporozoites in a buffer such as phosphate buffer, and inoculating chickens with this solution. Inoculation sites include the oral cavity, muscle, and cloaca, and it is particularly preferable to inoculate the cloaca. Specific methods for cloacal inoculation include dropping sporozoites into the anus of chickens or injecting them into the cloaca. The amount of sporozoites inoculated should be about 500 or more per chicken. By using the anti-coccidial agent according to the present invention, coccidiosis caused by Eimeria tenella can be almost completely prevented without causing side effects. Furthermore, the infection-preventing effect is very effective, for example, when day-old chicks are inoculated with the anti-coccidial agent of the present invention for a long period of 11 weeks or more. Next, the following examples are given to demonstrate the effects of the present invention. Example 1 Test method: Drop the following sample into the anus of 10 0-day-old chickens (broiler breed Hubbard). The chickens were then given 200,000 Eimeria per bird at 21 days of age.
Mature oocysts of Tenella are administered orally to infect coccidiosis. As a control, only infection was performed without inoculating the sample. On the 8th day after infection, the rats were observed for cecal lesions, the number of oocysts in the feces, the presence or absence of bloody stool, and the mortality rate. The results are shown in Table 1. The evaluation was performed according to the following method. Sample Invention: 10,000 Eimeria tenella sporozoites suspended in a PBS(-) solution. Comparative example 1: Eimeria tenella spocysite
5000 pieces (10,000 pieces as sporozoites) on PBS
(-) Suspended in a solution. Comparative example 2: Eimeria tenella oocyst 1250
(10,000 sporozoites) in PBS (-)
suspended in a solution. Comparative Example 3: The sample of the present invention (1) was heated at 60°C for 30 minutes. Comparative example 4: Eimeria tenella sporozoite 1
10,000 pieces were placed in a 10% formalin solution and left for 30 minutes, then this solution was centrifuged to remove the precipitate.
Suspended in PBS(-) solution. The preparation method of oocysts, sporocysts, and sporozoites used as samples is described in "J.Protozool." Volume 9.
(2) The following procedure was carried out based on the method described on pages 154 to 161 (1962). The feces, cecal tube wall, and cecal contents of chickens to which mature oocysts of Eimeria tenella were orally administered were collected and suspended in a 3% potassium dichromate solution.
The cells were then cultured at 28°C for 2 days. The obtained solution containing mature oocysts was centrifuged (3000 rpm,
10 minutes) and collect the precipitate. The precipitate is then washed with water and saturated brine is added. The solution is centrifuged (3000 rpm, 10 minutes), the supernatant is removed, the supernatant is diluted with water, the diluted solution is further centrifuged (1000 rpm, 3 minutes), and the precipitate is collected. This precipitate consists almost exclusively of oocysts (used in Comparative Example 2). the oocysts with water and 5
% sodium hypochlorite solution and left to stand for 15 minutes, centrifugation (2000 rpm, 3 minutes) is performed.
The precipitate obtained is washed with water and then ground in a homogenizer. This ground material consists mostly of sporocysts (used in Comparative Example 1). This sporocyst was treated with 5% chicken bile and 0.25% trypsin in PBS (−).
Add the solution and allow to digest in a 40°C warm bath for approximately 1.5 hours. The obtained digestive fluid is centrifuged to collect a precipitate, and this precipitate is washed with a PBS(-) solution. This consists mostly of sporozoites (used in the present invention). Evaluation method Cecal lesions: Indicates the degree of cecal atrophy and bleeding, and the amount of inflammatory exudate. : Severe: Moderate +: Mild ⊥: Very mild -: None Bloody stool: The severity is similar to that of cecal lesions. Number of oocysts in feces: represents the number of oocysts in 1 g of feces.

[Table] Example 2 Test method Ten chickens (used in Test Example 1) were inoculated with the sample shown below using the method shown below. Next, infection and observation were performed according to the method of Test Example 1. As a control, only infection was performed without inoculating the sample.
The results are shown in Table 2. The evaluation method is also the same as in Example 1. Sample and inoculation method Present invention (1): 10,000 sporozoites per chicken are inoculated into the anus of the chicken. Present invention (2): Chickens are orally inoculated with 10,000 sporozoites per chicken. Present invention (3): 10,000 sporozoites per chicken are injected into the pectoral muscle of the chicken. Comparative example: Chickens are orally inoculated with 1250 oocysts per chicken. Note that the method for preparing sporozoites and oocysts using the sample is similar to the method in Example 1.

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Claims (1)

[Claims] 1. An anti-coccidial agent containing Eimeria tenella sporozoites as an active ingredient. 2. A method for preventing coccidiosis in chickens, which comprises inoculating the cloaca of chickens with an anti-coccidial agent containing Eimeria tenella sporozoites as an active ingredient.